Glycoprotein Ib is homogeneously distributed on external and internal membranes of resting platelets

Abstract

Recent ultrastructural studies have suggested that Glycoprotein Ib (GPIb) has a different distribution on external (surface) versus internal (open canalicular system) membranes in resting discoid platelets. The differential distribution proposed for GPIb differs from that reported for the fibrinogen receptor, GPIIb-IIIa, and could have profound physiological significance when platelets are activated by surfaces. The present study explored the distribution of GPIb on external and internal membranes of resting platelets. Immunogold cytochemical techniques were applied to ultrathin cryosections of washed platelets. Polyclonal antibodies or mixtures of monoclonal antibodies (AP1 and 6D1) were used for labeling. To avoid the technical problem posed by limited accessibility of antigens located in very narrow portions of the open canalicular system (OCS) to antibodies, the same methods were applied to patients with giant platelets syndromes. The OCS of normal resting platelets was also dilated by exposure of platelets to hypertonic conditions or to cytochalasin-B, an agent that prevents assembly of actin, and, reportedly, movement of GPIb. Morphometric analysis revealed that rates of labeling on internal versus external membranes of giant platelets does not differ significantly (0.93 +/- 0.20), provided the OCS is sufficiently dilated. Platelets exposed to cytochalasin B (1.01 +/- 0.31) or to hypertonic conditions (0.96 +/- 0.20) revealed similar ratios for immunogold particles on external and internal membranes. Results of our study indicate that membranes of the exposed surface and lining OCS channels of resting platelets are continuous, identical structures and GPIb is homogeneously distributed on external and internal membranes.

Figure 1.

Thin section of a giant platelet from a patient with the May-Hegglin anomaly. An abundance of dilated channels making up the open canalicular system (OCS) fill the cytoplasm of the cell. Alpha granules (G) and occasional mitochondria (M) are prominent in the central region. Original magnification, ×18,800.

Figure 2.

Thin section of a giant platelet from the peripheral blood of a patient with Epstein’s syndrome. An extensive system of dilated OCS channels fills the cell matrix. In some areas the OCS channels are in close association with canaliculi of the dense tubular system forming membrane complexes (MC). Original magnification, ×22,000.

Figure 3.

Giant platelet from patient with May-Hegglin anomaly. The frozen thin section has been stained with a polyclonal antibody to the glycocalicin portion of GPIb and protein A coupled to 5-nm gold particles. Immunogold particles indicating sites of GPIb cover the exterior surface (S) of the large cell and line channels of the dilated OCS. Densities of labeling for immunogold detecting GPIb are comparable on the external surface or on membranes lining channels of the OCS. Original magnification, ×80,000.

Figure 4.

Cryosections of giant platelets from (A) a patient with Epstein’s syndrome (ES) and (B) a patient with Bernard Soulier syndrome (BSS). The cells were stained with the anti-glycocalicin antibody and protein A gold in the same manner as the cell in Figure 3 ▶ for the presence of GPIb. Gold particles indicating sites of GPIb cover the exposed surface (S) and membranes lining channels of the dilated OCS. There is no difference in the frequency of gold beads detecting GPIb on external or internal membranes of the giant ES cell. The specificity of the antiglycocalicin antibody for GPIb is indicated by the virtual absence of immunogold particles on and in the BSS platelet in B. Original magnifications, ×100,000 (A) and ×70,000 (B).

Figure 5.

Cryosection of normal platelet prepared after incubation with cytochalasin B (CB). The thin frozen section was incubated first with the monoclonal antibodies AP1 and 6D1 against GPIb, and then to anti-mouse IgG coupled to 10 nm gold. Immunogold particles indicating sites of GPIb cover the exterior surface (S) and membranes lining channels of the OCS. Tangential sections of the exposed surface may appear to have more gold particles, but there is no significant difference in the frequency of gold particles indicating GPIb on cross sections of external and internal membranes. Original magnification, ×46,000.

Figure 6.

Cryosection of a normal platelet incubated with cytochalasin B stained for GPIb in the same manner as the cell in Figure 5 ▶ . Immunogold beads detecting sites of GPIb are prominent on membranes of the cell surface (S) and lining the OCS. The receptors appear to have the same density on internal and external membranes. Original magnification, ×42,000.

Figure 7.

Cryosection of a larger normal platelet with a dilated OCS prepared after exposure to CB. The frozen thin section was stained with AP1, 6D1, and anti-mouse IgG coupled to 10 nm gold in the same manner as the cell in Figure 5 ▶ . Immunogold beads indicating sites of GPIb cover the external and internal membranes. The frequency of gold particles is essentially the same on the exposed surface (S) and membranes lining OCS channels. Original magnification, ×42,000.

Figure 8.

Cryosection of a normal platelet prepared for study after incubation under hypertonic conditions (see text). The frozen section was incubated first with API and 6DI and then with anti-mouse IgG coupled to 10 nm gold. Hypertonic salt solutions have caused dilatation of the OCS. Gold particles detecting sites of GPIb are evenly dispersed on the exposed surface (S) and membranes lining channels of the OCS. There is no significant variation in the frequency of GPIb on external and internal membranes. Original magnification, ×39,000.

Figure 9.

Frozen thin sections of a normal platelet exposed to hypertonic conditions before freezing. The cryosection has been stained for GPIb in the same manner as the cell in Figure 8 ▶ . Gold beads detecting sites of GPIb are evenly dispersed on the surface (S) membrane and linings of OCS channels. The variation in frequency of GPIb on the outer and interior membranes is not significant. Original magnification, ×33,000.

Figure 10.

Cyrosection of another normal cell prepared after exposure to hypertonic conditions. The frozen thin section was stained for GPIb in the same manner as the cells in Figures 8 and 9 ▶ ▶ . Gold particles indicating the presence of GPIb are dispersed evenly on the exposed surface (S) and in the OCS. Differences in gold particle frequency on outer and inner membranes are not apparent. Original magnification, ×42,000.

Figure 11.

Comparison of the number of gold particles on external (white bars) and internal membranes (dashed bars) of control, giant, cytochalasin B-treated, and hypertonically treated platelets. Densities of labeling are expressed in particles/μm. Statistical differences were observed for labeling on external versus internal membranes in control platelets (*P < 0.05). Differences were not observed under conditions where the open canalicular system was naturally or artificially dilated.